Glucose determination method employing orthotoluidine

ABSTRACT

AN IMPROVED ORTHO-TOLUIDINE REAGENT COMPOSITION FOR USE IN DETERMINATION OF GLUCOSE IN BIOLOGICAL FLUIDS COMPRISES SUFFICIENT OXALIC ACID OR POLYPHORPHORIC ACID TO PROVIDE A UNIFORM INTENSITY OF COLOR WHEN THE COMPOSITION IS REACTED WITH GLUCOSE, INDEPENDENTLY OF CONTAMINATION WITH TRACE AMOUNTS OF IRON.

United States Patent 3,711,250 GLUCOSE DETERMINATION METHOD EMPLOYENG ORTHOTOLUIDINE William S. Stavropoulos, Carmel, and Bernard J. Thiegs, Indianapolis, Ind., assignors to The Dow Chemical Company, Midland, Mich. No Drawing. Filed Nov. 1, 1971, Ser. No. 194,682 Int. Cl. G01n 33/16 US. Cl. 23-230 B 8 Claims ABSTRACT OF THE DISCLOSURE An improved ortho-toluidine reagent composition for use in determination of glucose in biological fluids comprises sufiicient oxalic acid or polyphosphoric acid to provide a uniform intensity of color when the composition is reacted with glucose, independently of contamination with trace amounts of iron.

BACKGROUND OF THE INVENTION The determination of glucose in biological fluids using an ortho-toluidine reagent composition is an accepted method in many hospital, industrial, reference and commercial clinical laboratories. The method is based on the color formed when glucose reacts with ortho-toluidine in glacial acetic acid during a short heating period. The intensity of the resulting blue-green color is measured photometrically. See Dubowski, Clin. Chem. 8; 215 (1962) and Hyvarinen et al., Clin. Chim. Acta 7; 140 (1962).

Conventional ortho-toluidine reagents generally contain from about to about grams per 100 milliliters of ortho-toluidine in glacial acetic acid and often contain a small amount of thiourea usually in a concentration of from about 0.1 to 1 gram per 100 milliliters, to prevent subsequent discoloration of the reagent. The reagents may also contain small amounts of hydrazine to provide uniformity in the formation of color with different lots of reagent composition.

The glucose determination is usually performed on a biological fluid such as serum, plasma or urine; however, Whole blood, cerebrospinal fluid, milk, and other biological fiuids may be analyzed. A predetermined volume of sample fluid, generally 0.100 milliliter, is mixed with a predetermined volume of ortho-toluidine reagent composition, generally from about 3 to about 5 milliliters, in a suitable container such as a colorimeter cuvette or test tube. The mixture is heated to develop color, typically in a heating block at 100 C. or boiling water bath for a predetermined period of time, usually from 7 to 10 minutes, and in the intensity of the resulting blue-green color is measured photometrically in a spectrophotometer or colorimeter at a wavelength from 590 to 625 millimicrons or nanometers, and compared with a standard glucose solution, calibration curve or the like.

The amount of color produced in the ortho-toluidine color reaction is drastically reduced by the presence of small amounts of iron. Iron may be present in varying amounts as a contaminant in the vials, cuvets, pipets or other glassware used in preparation of the reagent or sample, or it may be introduced as a contaminant in one of the ingredients of the reagent. It may be introduced in varying amounts from water used to wash or rinse labortory glassware. Variations in the degree of iron contamination can bring about undesirable variations in color intensity from lot to lot of reagent composition or from test to test. Both ferric and ferrous iron interfere with the color reaction, and due to the acidic nature of the reagent composition, contact with metallic iron can also result in interference. The interference resulting from iron contamination is manifested by decreased final absorbance. Amounts of iron on the order of l to 2 parts per million have been observed to reduce final absorbance by as much as 30 to 50 percent, so that the analytical results are correspondingly decreased. Such interference, if undetected or uncorrected, can thus pose a significant risk of misdiagnosis based on an erroneously low result.

There is a need for a simple improvement in orthotoluidine reagents which provides for regulation of the color developed with glucose to obtain uniform absorbance from batch to batch, from sample to sample, or from test to test independently of iron contamination.

BRIEF SUMMARY OF THE INVENTION This invention relates to an improvement in orthotoluidine reagent compositions used for determination of the glucose concentration in biological fluids. The invention gives a uniform intensity of color when reacted with a specified quantity of glucose, independently of contamination with small amounts of iron.

It has now been found that the intensity of color developed in glucose determinations carried out with a conventional ortho-toluidine reagent composition can be made uniform in the presence of iron in the concentrations ordinarily encountered in iron contamination (e.g., from about zero to about three, up to about ten parts by weight of iron per million parts by weight of reagent composition). Such uniformity can be provided by the addition of a minor amount of oxalic acid, polyphosphoric acid or a mixture thereof to the reagent composition prior to developing color, and preferably prior to mixing the reagent composition with glucose.

The addition to the reagent composition of oxalic or polyphosphoric acid in an amount sufiicient to prevent interference by trace amounts of iron provides a reagent composition which gives a consistent uniform absorbance and intensity of color independently of the presence or absence of trace amounts of iron in the ultimate color reaction mixture. Interference is prevented independently of the valence state (ferrous or ferric ion) of the iron contaminant.

In formulating and using ortho-toluidine reagents according to the invention, the reagent composition can be formulated with the same ortho-toluidine and glacial acetic acid and with thiourea and hydrazine and other ingredients (if desired) in the same proportions as reagents typically employed, and the glucose determination can be carried out in the identical procedures, with the same proportions of reagent composition and glucose standard or sample as have been heretofore employed, with the sole modification being the addition to the reagent composition of sufiicient of oxalic acid, polyphosphoric acid or a mixture thereof to provide a substantially uniform final absorbance after maximum color development over a range of iron concentrations of from about zero to about one, to about three to about ten parts of iron per million parts of reagent composition. The oxalic acid brpolyphosphoric acid ingredient can be added to in formulating the reagent, the reagent composition at any time prior to developing color, for example, immediately before adding a sample or a glucose standard solution, or after such addition but prior to heating to develop color. The acid ingredient is preferably added to the other ingredients by parts per million to the maximum expected, generally about 10 parts per million. In general, the final absorbance should appear statistically uniform at the 95 percent confidence level, independently of the presence or absence of iron, the resulting composition will be well protected from interference by iron contamination.

The oxalic or polyphosphoric acidingredient is generally employed in amounts of from about 0.01 to about 0.05 to about 0.1 to about 0.5 to about 1 gram of acid per 100 milliliters of reagent composition, to saturation of the composition with the acid. It is generally unnecessary to employ the acid ingredient in amounts greater than about one gram per 100 milliliters, and amounts of from about 0.05 to about 0.5 gram per 100 milliliters of final reagent composition are preferred. It is essential, however, that one of the named acid ingredients be employed. Other acids, such as ethylenedinitrilotetraacetic acid, malonic acid, succinic acid, adipic acid, maleic acid, phthalic acid, citric acid and phosphoric acid are ineifective in preventing the interference caused by iron contamination.

In practice the relative proportions of the other ingredients in the reagent composition, and the parameters of the analytical procedure such as proportions of reagent composition and standard or sample, heating time and temperature for color development, wavelength of light at which color intensity is measured, etc. are determined in accordance with known procedures. The ortho-toluidine reagent composition and the glucose determination procedure are conventional in the determination of glucose by reaction with ortho-toluidine as the essential color forming reactant. Preferably the reagent compositioribontains from about 5 to about grams of ortho-toluidine per 100 milliliters of ultimate composition and from about 0.1 to about 1 gram of thiourea per 100 milliliters, the remainder of the composition being glacial acetic acid with the required amount of oxalic or polyphosphoric acid and optionally a small amount of hydrazine added to control the'desired final absorbance. A preferred composition contains about 9 grams of ortho-toluidine, 0.5 gram thiourea, 60 to 200 micromoles of hydrazine, and from about 0.01 to 0.5 gram of oxalic or polyphosphoric acid, in glacial acetic acid to obtain a final volume of 100 milliliters. Such reagent composition is preferably employed in glucose determination by mixing one part by volume of a biological fluid sample or a glucosestandard solution with about 30 to 50 parts by volume of reagent composition, heating the mixture in a tube in a heating block or water bath at a temperature of about 100 C. for about 7 minutes, then cooling in a cold water bath (temperature of water bath below 25 C.) for about three minutes before measuring color intensity with light having a wavelength of 590 to 625 millimicrons (nanometers). With such a reagent composition and method, excellent uniformity of final absorbance is obtained independently of iron contamination.

DESCRIPTION OF PREFERRED EMBODIMENTS The following examples are illustrative of the invention.

7 Example 1 A series of ortho-toluidine reagent compositions is prepared by mixing together the following ingredients in the following proportions:

Reagent A:

" o-Toluidine 1 Q. granis 9.0 Thiourea 0.5 Glacial acetic acid, q.s. to 100 milliliters.

Reagent B: I

' o-Toluidine 1 grams 9.0 Thiourea do 0.5 200 ppm. Fe++ solution do 1.0

Glacial acetic acid, q.s. to 100 milliliters.

Reagent C:

o-Toluidine 1 grams 9.0 Thiourea do 0.5 200 p.p.m. Fe++ solution milliliter 1.0 5 Oxalic acid gram 0.1

Glacial acetic acid, q.s. to 100 milliliters. Reagent D:

o-Toluidine 1 "grams" 9.0 Thiourea do 0.5 10 Oxalic acid do 0.1 Distilled water milliliter 1.0 Glacial acetic acid, q.s. to 100 milliliters.

Containing a minor amount of hydrazine (less than 0.1 percent).

Solution of 140 milligrams of FQ(NH4)2(SO4)26H2O in 100 milliliters of 0.1 normal sulfuric acid. 7

Each reagent is tested in duplicate with 0.100 milliliters of an aqueous glucose standard solution containing 100 milligrams of glucose per 100 milliliters. Five milliliters of reagent composition are transferred to each of three vials and one vial used as a reagent blank. One hundred microliters of the glucose standard is added to each of the two remaining vials. These two vials are capped and the contents mixed well by shaking for about 5 seconds. The vials are then placed in a heating block at 100 C. for 7 minutes, after which they are then removed and cooled in a cold water bath for 3 minutes. The intensity of the blue-green color given by the glucose standard is then measured at 595 nonometers using a photoelectric colorimeter. (The instrument is previously. adjusted to zero absorbance using the untreated vial of reagent.) The results are set out below.

Average Absorbance absorbanee' Reagent composition X100 100 Reagent A. (Control) 31. 9

Reagent B (2 p.p.m. Fe++ iron) 17. 3

Reagent C (0.1% oxalic acid plus 2 ppm. 32 D Feiron) 5 31. 8 40 Reagent D (0.1% oxalic acid) p 32. 3

These data indicate the severe decrease in absorbance resulting from the relatively slight degree of iron contamination, and further show the excellent correction pro- 48 vided by the addition of oxalic acid.

7 Example 2 In a similar operation, for reagent compositions E, 50 F, G and H are prepared and tested. The composition of the reagents and the average absorbances obtained are set out below:

Average absorbfirlige Reagent E:

o-Toluidine 9.0% w./v Thiourea 0.5% wJv... 33.9 Glacial acetic acid- Q.s. to 100% by vol Reagent F:

o-Toluidine l 9.0% w./v Thiourea 0.5% w./v 15 6 2,000 ppm. Fe+++ iron solution 0.17 v./v Glacial acetic acid Q,.s. to 100% by vol.. Reagent G:

o-To1uidine 9.0% wJv- Thiourea 0.5% w.[v Polyphosphoric acid 0.1% w.[ 33. 5 2,000 ppm. Fe++ iron solution 0.1% vJv Glacial acetic acid Q.s. to 100% by voi Reagent H:

' o-"Ioluidine 9.0% w.lv

Thiourea 0.5% w.lv 33 9 Poiyphosphoric acid .1% w./v

' Glacial acetic acid Q.s. to 100% by VOL.

7O 1 Orthotoluidine containing a minor amount (less than 0.1 percent) of hydrazine.

1 Solution of 0.97 gram of FeCh-GHQO in 100 milliliters of glacial acetic acid. The final concentration of iron in the reagent corresponds to 2 parts per million.

' 3 Polyphosphoric acid (11 1 4013 approx.) containing 82-84% P205.

No'rE.-% wJv. represents percentage in weight by volume correspending to grams per 100 millilters; inlv." represents percentage by volume.

Example 3 xalic acid (percent (w./v.)

Average absorbance X100 Iron (p.p.m.):

In other operations, similar uniform results are obtained with iron concentrations of parts per million.

It will be apparent to those skilled in the art that numerous variations can be made in the use of the invention, for example, by varying the concentration of ortho-toluidine, and by varying the amounts and the nature of other ingredients in adapting to ortho-toluidine color reaction to particular analytical systems, while utilizing the invention to protect against the effects of iron contamination.

What is claimed is:

1. In a method for determination of glucose comprising the steps of mixing together predetermined quantities of a color forming reagent and a sample composition comprising glucose and developing color in the mixture resulting from both said mixing steps;

the improvement which comprises the step of adding an acid selected from oxalic acid, polyphosphoric acid and mixtures thereof to the color reagent composition prior to the step of developing color, the acid being added in an amount sufficient to provide a uniform intensity of color which is independent of the presence of trace amounts of iron in said mixture.

2. The method of claim 1 wherein the color reagent composition is prepared by mixing together ortho-toluidine, thiourea, hydrazine and glacial acetic acid in predetermined amounts, and wherein said acid is mixed with the color reagent composition prior to mixing of the reagent composition with the sample.

3. The method of claim 1 wherein said acid is employed in an amount sutficient to provide from about 0.01 gram of said acid per milliliters of color reagent composition, to saturation of the color reagent composition.

4. The method of claim 1 wherein said acid is added in an amount of from about 0.05 to about 0.5 gram of said acid per 100 milliliters of reagent composition.

5. The method of claim 1 wherein the reagent composition is contaminated with iron.

6. In a reagent composition useful for determination of glucose by measuring the intensity of color produced by the reaction of glucose with ortho-toluidine, and comprising a solution of ortho-toluidine in glacial acetic acid; the improvement wherein the reagent composition further comprises from about 0.01 gram per 100 milliliters to about saturation of an acid ingredient selected from oxalic acid, polyphosphoric acid and mixtures thereof, whereby said reagent composition is protected from interference by iron contamination.

7. The reagent composition of claim 6 wherein the composition contains from about 0.05 to about 0.5 gram of said acid ingredient per 100 milliliters of ultimate reagent composition.

8. The reagent composition of claim 6 wherein the reagent is contaminated with iron.

References Cited UNITED STATES PATENTS 3,615,228 10/1971 Thiegs et a1. 23-230 B MORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner U.S. Cl. X.R. 252408 

